Assembly of DNA into functional genetic elements is a fundamental aspect of molecular genetics. DNA assembly methods generally require the generation of DNA fragments having defined, compatible ends that are suitable for subsequent joining, or ligation.
Traditionally, compatible end of DNA fragments are generated by the use of restriction enzymes which can be joined together by a DNA ligase. If restriction enzymes leaving protruding ends are used, directional joining of segments containing matching overlaps can be achieved. Traditional cloning methods have several disadvantages. First, due to unproductive side reactions (either circularization of segments by self-ligation or formation of runway concatamers), traditional ligation methods are not very efficient, with the assembly of more than 3 compatible DNA segments into circular vectors especially inefficient. This method also relies on presence of unique restriction sites at particular locations in the precursor DNA molecules from which the DNA fragments to be joined are derived. These sites might not be available, especially if large fragments are combined. In addition, most restriction sites require the presence of a specific sequence at the end of the processed molecule which lead to “scars” after ligations, often interfering with the ultimate goal of seamless assemblies.
To overcome the ligation junction problem, some methods employ the use of type II restriction enzymes that cleave outside of their recognition sequence. These recognition sequences are added to the ends of the combined fragments, usually by PCR. If the restriction enzyme generates staggered ends, the resulting fragments can be combined directionally resulting in “seamless” assemblies. This approach is the basis of the “golden gate” cloning approach, where suitable restriction sites internal to the cloned fragments can be protected from cleavage by introducing methylations during the PCR amplification of the cloned segments (see e.g., U.S. Pat. No. 6,261,797).
In an alternative approach, DNA fragments can be combined using matching “long” overlapping sequences at their ends. If complementary single stranded ends are generated, typically by specifically degrading one of the DNA strands by either a 5′ or 3′ exonuclease, the combined segments can be annealed to each other. The annealed segments, usually with overlap lengths of 12-13 nucleotides, are introduced into bacterial host cells where the gaps in the annealed DNA are repaired and sealed by the host repair systems. This approach is generally referred to as “ligation independent cloning” or LIC cloning (see e.g., Haun et al., “Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors.” BioTechniques 13 (4): 515-8 (1992)). In a variant of this approach known as “Gibson assembly” (see e.g., Gibson et al., “Enzymatic assembly of DNA molecules up to several hundred kilobases.” Nature Methods 6 (5): 343-345 (2009)), matching 3′-protruding ends are generated using a 5′-exonuclease. The annealing products are then repaired using a non-strand-displacing DNA polymerase and a ligase to seal potential gaps, thus recreating in vitro some of the host repair processes utilized during LIC-cloning.
A different, overlap dependent assembly method is based on assembly of DNA segments with overlapping ends using a thermal cycling protocol. This process referred to a CPEC assembly (see e.g., Quan, J., and Tian, J. “Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways.” PLoS ONE 4(7): e6441 (2009)) resembles a PCR reaction where the overlaps of the DNA sequences act as primer. The general drawback of this approach is that the amplification process never results in a closed circular product as is required for most bacterial plasmids. When circular permutated overlapping ends are utilized, the typical products are long concatemers. Formation of circular plasmids most likely results from overlapping, off-set intermediates with protruding single stranded ends that are repaired by the host cell. However, due to their structure, these are not easily taken up by the bacterial host, especially when large construct are assembled. Another disadvantage is that larger constructs also require very long cycling protocols.
As such, there exists a need for high efficiency DNA assembly methods and compositions for performing such methods.